Multi-photon microscopy in cardiovascular research.

Multiphoton laser scanning microscopy has proven profound value for ex vivo 3D histology and in vivo imaging of motionless tissue. The development of triggering systems and fast imaging methods, combined with advanced preparation procedures solved the challenging task of intravital imaging of the fast pulsating heart and major arteries in animals and further increased the popularity of intravital multiphoton imaging in cardiovascular research. This review article will highlight the potential of multiphoton microscopy for the visualization and characterization of dynamical and structural processes involved in cardiac and vascular diseases, both in an ex vivo and an intravital animal setting. Examples will be given how multiphoton microscopy can be applied to imaging of atherosclerotic plaque development and progression at subcellular level as well as to intravital imaging of inflammatory processes in the heart. In addition to highlighting the potential of multiphoton microscopy in preclinical cardiovascular research, we will discuss how this tool and its applications may be clinically translated to support disease diagnosis and therapy in patients.

Healthy, wealthy and wise: the benefits of chemical research.

Chemistry is the great enabling subject that is central to progress in solving global problems. In today's world, these problems are as vital, as new energy sources, food, clean water and healthcare. David Philips presents some current research in these areas as well as his own work in the field of targeted photodynamic therapy of cancer.

[Frontiers in Live Bone Imaging Researches. Two-Photon Excitation Microscopy, principles and technologies].

The "two photon absorption" phenomenon had been predicted by the American Physicist, Maria Ghöppert-Mayer in 1931. Denk and Webb group had proved it in 1990 and the first product had been launched in the market in 1996. But ever since the product became available, the number of users are not increased. Moreover, the system had been too difficult to use and the system sometimes stay not working in labs. But recently, the new easier-to-use products are released and the ultra short pulse IR laser became stable. And its applications are extending from neuro-science to oncology or immunology fields. Due to these reasons, the shipment of multi-photon microscope in Japan in 2013 is approximately 40 units which is 3 times bigger than in 2010. In this paper, I would like to discuss the principles of two-photon microscopy and some of the new technologies for the higher signal capture efficiency.

Update of technologies for examining the stratum corneum at the molecular level.

Understanding the molecular organization of the stratum corneum is still an outstanding problem, despite being both fundamentally and clinically significant. There is a need to develop methodology that yields molecular-level resolution of the stratum corneum components in their native state, without introducing artefacts. We outline here the recent success of cryo-electron microscopy of vitreous sections (CEMOVIS) combined with electron microscopy simulation to elucidate the molecular organization of the stratum corneum in its near-native state. Furthermore, some emerging technologies for studying the physical properties and dynamic behaviour of native stratum corneum at the molecular level are briefly reviewed. These encompass multiphoton microscopy (MPM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR) and PeakForce tapping-mode atomic force microscopy combined with frequency-modulation Kelvin probe force microscopy (KPFM). CEMOVIS combined with electron microscopy simulation allows for molecular structure determination in situ in native stratum corneum, while MPM allows probing of the stratum corneum local physicochemical properties such as fluorophore diffusion coefficients, water content and pH. PTssNMR allows for evaluation of the molecular mobility of stratum corneum keratin and lipid components, and PeakForce KPFM allows for analysis of the local nanomechanical properties of stratum corneum. These emerging techno-logies may contribute to a molecular-level understanding of stratum corneum structure and function in vivo.

The first decade of using multiphoton microscopy for high-power kidney imaging.

In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans.

Special Section Guest Editorial: Thirty Years of Multiphoton Microscopy in the Biomedical Sciences.

JBO guest editors introduce the Special Section Celebrating Thirty Years of Multiphoton Microscopy in the Biomedical Sciences.

Multiphoton microscopy: a personal historical review, with some future predictions.

The historical development of multiphoton microscopy is described, starting with a review of two-photon absorption, and including two- and three-photon fluorescence microscopies, and second- and third-harmonic generation microscopies. The effects of pulse length on signal strength and breakdown are considered. Different contrast mechanisms, including use of nanoparticles, are discussed. Two new promising techniques that can be applied to multiphoton microscopy are described.

Nanoparticles for optical molecular imaging of atherosclerosis.

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

Interphase chromosome-specific multicolor banding (ICS-MCB): a new tool for analysis of interphase chromosomes in their integrity.

Biomedical research of interphase chromosomes in their integrity is hindered by technical limitations. We introduce a technology using microdissection-based engineering of DNA probes and fluorescence multicolor chromosome banding that allows studying interphase chromosome organization, numbers and rearrangements in somatic cells.

Nonlinear magic: multiphoton microscopy in the biosciences.

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

High-resolution large-scale mosaic imaging using multiphoton microscopy to characterize transgenic mouse models of human neurological disorders.

The thorough characterization of transgenic mouse models of human central nervous system diseases is a necessary step in realizing the full benefit of using animal models to investigate disease processes and potential therapeutics. Because of the labor- and resource-intensive nature of high-resolution imaging, detailed investigation of possible structural or biochemical alterations in brain sections has typically focused on specific regions of interest as determined by the researcher a priori. For example, Parkinson's disease researchers often focus imaging on regions of the brain expected to exhibit pathology such as the substantia nigra and striatum. Because of limitations in acquiring and storing high-resolution imaging data, additional data contained in the specimen is not usually acquired or disseminated/reported to the research community. Here we present a method of imaging large regions of brain at close to the resolution limit of light microscopy using a mosaic imaging technique in conjunction with multiphoton microscopy. These maps are being used to characterize several genetically modified animal models of neurological disease by filling the information "gap" among techniques such as magnetic resonance imaging and electron microscopic analysis.

Technologies for imaging neural activity in large volumes.

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Conventional microscopy collects data from individual planes and cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point-spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for processing and analyzing volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics and helping elucidate how brain regions work in concert to support behavior.

Alzheimer's disease: what multiphoton microscopy teaches us.

A definitive diagnosis of Alzheimer's disease depends on postmortem analysis of brain tissue bearing the pathological hallmarks of the disease: plaques and tangles. Imaging techniques that allow visualization and characterization of these lesions in living animals permit a better understanding of the pathogenesis of the disease as well as paradigms for preventing or reversing the deposits. Multiphoton microscopy uses near infrared light that is benign to living tissue and penetrates more deeply than visible or UV light, permitting high-resolution imaging of these microscopic structures deep within the cortex of living transgenic mice over time. This in vivo imaging approach allows direct examination of the natural history of plaques and evaluation of antiplaque therapeutics in mouse models of the disease.

Applications of multi-photon microscopy in cell physiology.

Owing to its many optical and physical advantages for fluorescence excitation, multi-photon microscopy has found a wide range of uses in biology, both in structural and functional studies. In this review we highlight various applications of this technique in different fields of cell physiology and biophysical research. This includes studies on second messenger and ionic signals, on cellular metabolism as well as on genetically engineered probes and indicators. In addition, this techniques has been successfully applied for diffraction-limited photolysis of caged compounds. We also point out some of the problems that were encountered along the still rapidly evolving path of this technique, and draw attention to some of the ongoing developments that will further extend and improve the usefulness of multi-photon excitation, such as fluoresce life-time imaging (FLIM), fluorescence resonance energy transfer (FRET) and entangled photon microscopy approaches.

Three photons are better than two.

Three-photon microscopy was suggested in the 1990s, but laser technology at the time was just not up to the challenge. Lauren Ware explores how recent technology advances are bringing three-photon microscopy back into focus.

Genetically encoded neural activity indicators.

Recording activity from identified populations of neurons is a central goal of neuroscience. Changes in membrane depolarization, particularly action potentials, are the most important features of neural physiology to extract, although ions, neurotransmitters, neuromodulators, second messengers, and the activation state of specific proteins are also crucial. Modern fluorescence microscopy provides the basis for such activity mapping, through multi-photon imaging and other optical schemes. Probes remain the rate-limiting step for progress in this field: they should be bright and photostable, and ideally come in multiple colors. Only protein-based reagents permit chronic imaging from genetically specified cells. Here we review recent progress in the design, optimization and deployment of genetically encoded indicators for calcium ions (a proxy for action potentials), membrane potential, and neurotransmitters. We highlight seminal experiments, and present an outlook for future progress.